Tetanus toxin elicits an electrophysiologic response in primary neuronal cultures similar to that observed in the nervous system in situ. A biochemical assay for neurotransmitter release was developed in order to define the phases of toxin action corresponding to the physiologic response. Excitatory and inhibitory neurotransmitter pools were radiolabeled, and transmitter release was assayed at various times after toxin exposure. Relative to control cultures, release of the inhibitory neurotransmitter, glycine, was unaffected during the latent period, and was inhibited during the phases of convulsant activity and electrical quiescence. Release of the excitatory neurotransmitter, glutamate, was unaffected during the latent period, slightly increased during convulsant activity, and inhibited during quiescence. These patterns of release parallel increases and decreases in the frequency of recorded excitatory and inhibitory postsynaptic potentials, and indicate that the phase of convulsant activity can be defined by differential transmitter release and electrical quiescence, by inhibition of transmitter release. Electron microscopy of synaptic terminals showed progressively fewer structures associated with synaptic vesicle release and recycling, and increased numbers of synaptic vesicles adjacent to release sites, with time of toxin exposure. These results indicate that the final toxin-induced blockade of synaptic transmission appears related to an inability to synaptic vesicles to fuse with the presynaptic release site. At the time when synaptic transmission between mature (i.e., more than four weeks in culture) spinal cord neurons is blocked by tetanus toxin, the synaptic connection between sensory and spinal cord neurons is not affected. Whereas toxin bound to the recorded spinal cord neurons can be visualized immunohistochemically, the sensory neurons appear unstained. Thus, the failure to block the sensory-spinal cord neuron connection may be related to the inability of toxin to bind to mature sensory neurons.